Reverse transcriptases are multi-functional enzymes with three enzymatic activities including RNA- and DNA-dependent DNA polymerization activity, and an RNaseH activity that catalyzes the cleavage of RNA in RNA-DNA hybrids. Mutants of reverse transcriptases have disabled the RNaseH moiety to prevent unintended damage to the mRNA. These enzymes that synthesize complementary DNA (cDNA) using mRNA as a template were first identified in RNA viruses. Subsequently, reverse transcriptases were isolated and purified directly from virus particles, cells or tissues. (e.g., see Kacian et al., 1971, Biochim. Biophys. Acta 46: 365-83; Yang et al., 1972, Biochem. Biophys. Res. Comm. 47: 505-11; Gerard et al., 1975, J. Virol. 15: 785-97; Liu et al., 1977, Arch. Virol. 55 187-200; Kato et al., 1984, J. Virol. Methods 9: 325-39; Luke et al., 1990, Biochem. 29: 1764-69 and Le Grice et al., 1991, J. Virol. 65: 7004-07). More recently, mutants and fusion proteins have been created in the quest for improved properties such as thermostability, fidelity and activity.
Copying RNA can be inhibited by the presence of RNA secondary structure which can stall cDNA synthesis resulting in truncated cDNA molecules. The formation of secondary structure can be avoided at higher temperature. While this also reduces non-specific priming and thereby increases reverse transcriptase fidelity, length and yield of cDNA. However, RNA integrity can be compromised by lower enzyme activity at elevated temperatures. Further improvements are desirable to obtain optimum performance of the enzymes in library synthesis and NextGen sequencing.